Re-evaluation of HeLa, HeLa S3, and HEp-2 karyotypes

In contrast to earlier reports, this study on HeLa, HeLa S3, and HEp-2 revealed that karyotypes of each cell line are characterized by a consistent marker chromosome composition and a constant number of copies for both normal and marker chromosomes. Based on these chromosome fingerprints and an analysis of 50 metaphases, the modal karyotype of each cell line was defined. Each modal karyotype had the composite content of the previously reported karyotypes of the same cell line, and, generally, the former had the same or a higher number of copies per chromosome than the latter. This modal karyotype can be used as a standard to identify and further individualize both the cell line itself and a subline within that cell line. We have also found that many cells within each cell line have the same karyotype. Portions of numerical data are compiled in a chart format by which the extent of chromosome differences between cultures can readily be compared. Also discussed in brief are characteristic chromosome changes that may help distinguish clonally derived cell lines from lines derived by cross-contamination.     

Mitochondrial Ribosomes in HeLa Cells

HeLa cell mitochondria contain 60S ribosomes which seem to consist of subunits of 45S and 35S particles. The 16S and 12S RNA components are coded by mitochondrial DNA.     

Electrolyte Metabolism in HeLa Cells

Methods have been developed to study cellular Na, K, and Cl concentrations in HeLa cells. Cell [Na] and [K] are functions of the age of the culture. As the culture grows [K], expressed in mmols/liter cell H₂O, rises from an initial value of 121 to a peak of 206 at about 4 days, and thereafter falls until it has almost returned to the initial value by the 9th day. [Na] falls as [K] rises, but there is no fixed relationship between the cellular concentrations of the two cations. There is, however, a correlation between generation time and cellular [K]. Measurements of net K uptake and net Na extrusion were carried out during 1 hour incubation at 37°C of low K cells. Both net K uptake and net Na extrusion took place against chemical concentration gradients, so that at least one transport system must be active; if the Cl distribution is passive both net K uptake and net Na extrusion are active. Studies with inhibitors of respiration and glycolysis lead to the conclusion that respiration is not required for these net transports, which appear to derive their energy from glycolytic sources.     

Ribosomal proteins of HeLa cells

Ribosomal proteins from HeLa cells were analyzed by two-dimensional polyacrylamide gel electrophoresis (Kaltschmidt-Wittmann) and dodecylsulfate polyacrylamide gel electrophoresis (Laemmli). 35 proteins are associated with the small ribosomal subunit and 47 proteins with the large ribosomal subunit. The HeLa ribosomal proteins S6, S32, L40b,c, L41 and L42 are phosphorylated in vivo and in vitro. Minor differences between HeLa and rat liver ribosomal proteins were revealed by their direct coelectrophoresis.     

Purification of a HeLa cell high molecular weight action binding protein and its identification in HeLa cell plasma membrane ghosts and intact HeLa cells

The high molecular weight protein (HMWP) which was previously observed to be a major component of the actin based gels formed by incubating cytoplasmic extracts of HeLa cells at 25 degrees C [Weihing, R. R. (1977) J. Cell Biol. 75, 95-103] has now been purified by gel filtration of 0.6 M KCl extracts of precipitated gels. A few hundred micrograms of HMWP, which is about 90% pure, can be isolated from 4 X 10(9) cells. HMWP can gel muscle actin and cross-link it into filament bundles. Its subunit molecular weight is 250 0000, its Stokes radius is 125 A, and its sedimentation coefficient is 9 S. A native molecular weight of 480 000 was calculated by using the latter two parameters, and therefore the native molecule is a dimer. Its amino acid analysis is nearly indistinguishable from that of macrophage actin binding protein and of mammalian and avian filamins. All of these findings indicate that HMWP is homologous to the latter proteins. However, HeLa cell HMWP and avian filamin must differ in their primary sequences because their partial peptide maps are distinct and because an antiserum against HMWP reacts only weakly with filamin. For studies on the intracellular location of HMWP, a goat antiserum against purified HMWP was prepared and characterized and then used to localize HMWP in suspension grown cells. The technique of immunoblotting revealed that the antiserum reacted virtually exclusively with the high molecular weight polypeptide that comigrates with HMWP in cell lysates and in ZnCl2-stabilized plasma membrane ghosts prepared from HeLa cells [Gruenstein, E., Rich, A., & Weihing, R. R. (1975) J. Cell Biol. 64, 223-234] and that it did not react with rabbit myosin heavy chain, microtubule proteins (MAPS and tubulin) from HeLa cells and calf brain, or the proteins of human erythrocyte ghosts including spectrin. Suspension-grown cells which were stained with the antiserum by the technique of indirect immunofluorescence showed bright fluorescence at the rim of the cells and less intense generalized fluorescence. If preimmune serum or immune serum treated with HMWP was substituted for the immune serum, then staining at the rim was not observed, but the generalized fluorescence was only slightly reduced; unpermeabilized cells were not stained. These results indicate that HMWP is a component of the cortical cytoplasm of HeLa cells. Possible functions of cortical HMWP are discussed briefly.     

UV-Induced Apoptosis in Resistant HeLa Cells

Recently, apoptosis (genetically programmed cell death) induced by UV hasbeen documented in some cell culture models. However, the significance ofapoptosis in UV-induced cytotoxicity and resistance is uncertain. In thisstudy, we investigated the induction of apoptosis in HeLa cells and itsrole in acquired UV-resistance. The membrane receptor Fas was induced toassembly, and its immediate downstream target, caspase-8, was induced byUV in a dose- and time-dependent manner. Caspase-10, another possiblecandidate for forming the death-inducing signaling complex with Fas, wasalso activated in a dose- and time-dependent manner. There was significantactivation of caspase 9, 3 and 2 by UV. The apoptotic pathways appeared tobe normal in acquired UV-resistant HeLa cells. In addition, there was a UVdose-dependent induction of chromatin condensation in both parental andUV-resistant cells. However, resistant cells displayed significant reductionin chromatin condensation at lower doses. Inhibition of caspase-3 activation byspecific inhibitor significantly reduced the chromatin condensation in bothcell types, and unexpectedly, the difference between the two cell lines wascompletely eradicated, suggesting that the caspase-3 pathway plays asignificant role in reducing apoptosis in resistant cells. The resultsindicate that UV induces apoptosis by direct activation of apoptoticproteins in HeLa and resistant cells. Although resistant cells displayedpartial inhibition of UV-induced apoptosis through the caspase-3 pathway,there was no consistent difference in the activation of this and relatedcaspase-9 caspases compared to parental HeLa cells.     

The biotin requirement of HeLa cells

We have examined the effect of alterations in the biotin content of the medium on the growth, viability, biotin content, and the activities of biotin-dependent and biotin-independent enzymes of the HeLa cells. The inclusion in the growth medium of avidin, which almost irreversibly binds with biotin (K_d, 10^?15 M), results in an increase in cellular biotin content and biotin enzyme activity over that seen when the cells are grown in a biotin-depleted medium. The addition of avidin-bound biotin to the growth medium led to a forty-fold increase in cellular biotin when compared to the inclusion of an equivalent amount of free biotin in the medium. HeLa cells are able to internalize avidin-bound biotin. Biotin is released from this complex to function as the prosthetic group of biotin enzymes. HeLa cells do have a nutritional requirement for biotin.     

RNA synthesis in permeable HeLa cells

The equilibria and kinetics are reported for the partial reactions of the catalytic cycle of the Ca²⁺ ionophore X537A in phospholipid vesicles. The analysis is based on the study of the behavior of the ionophore's intrinsic fluorescence in fluorescence lifetime, stopped-flow, temperature, and conventional steady-state fluorescence experiments. Binding to dimyristoyl phosphatidylcholine vesicles gives rise to an enhancement of the fluorescence. At the pH of study (7.4) this involves the singly negatively charged form (X^?). Complexation of the membrane-bound form (X_m^?) by monovalent (M⁺) or divalent (M²⁺) cations to give 1:1 (M-X)_m and (M-X)_m⁺ complexes, respectively, gives rise to a further fluorescence enhancement. No evidence could be found for stoichiometries other than 1:1 in the equilibrium experiments. The fluorescence of X537A in the presence of phosphatidic acid vesicles or phosphatidylcholine/ phosphatidylethanolamine or phosphatidylcholine/cholesterol mixtures is much smaller than for pure phosphatidylcholine. Fluorescence lifetime experiments show that this is due to a reduction in binding rather than a reduction of the quantum yield of the bound species. Fluorescence decay profiles from the above-mentioned membranes showed two exponential components indicating that there were two fluorescent species. The shorter-lived species had a lifetime of 3–5 ns and accounted for 80–90% of the membrane-bound ionophore. The longerlived species (9–13 ns) was estimated to account for the remaining 10–20%. This species enjoys a higher degree of hydrophobic shielding than the shorter-lived species. Possible interpretations in terms of the ionophore orientation in the membrane are discussed. Temperature-jump experiments show that the binding rate of the ionophore is fast. The binding and dissociation rate constants were ca. 2 × 10⁷m (PC)^?1 s^?1 and 2 × 10³ s^?1, respectively. Stopped-flow experiments gave evidence for a slower “insertion” process with a ca. 10-ms half-time. Analysis shows that this process is capable of transport of (K-X) across the membrane with a rate constant ≤ 69^?1. In the presence of divalent cations a slower process involving transport of M²⁺-ionophore complexes across the membrane can be observed. The dependence of the rate on the total ionophore concentration indicates that the transported species is a neutral (M-X₂) complex. The lower limit for the rate constant for transport of the (Ca-X₂) complex is 35 s^?1. The divalent cation specificity of the overall reaction was shown to be Mg²⁺ ? Ca² < Sr²⁺ < Ba²⁺. The rates of the overall transport at low ionophore concentration are limited by the equilibrium constant for formation of the (M-X₂)_m complex from the (M-X)_m⁺ complex.